Project Summary/Abstract The neurobiology of cocaine?s rewarding effects has been extensively characterized, but relatively little is known about the pathways and processes that underlie cocaine?s aversive effects. Aversive responses to cocaine are significant because they may modulate both the initial acquisition of drug-seeking, and later behaviors such as relapse in experienced animals, and may underlie individual differences in susceptibility to cocaine addiction. Prior work from our lab and others showed that activation of the rostromedial tegmental nucleus (RMTg) by cocaine critically drives conditioned avoidance responses to cocaine, and that some of this cocaine-induced RMTg activation is due to glutamatergic inputs from the lateral habenula (LHb). However, the LHb only explains a portion of this effect, and roughly half of the activation is not dependent on the LHb, and arises from unknown mechanisms. Using RNAseq I made the unexpected discovery that the RMTg expresses robust and enriched levels of mRNA for the serotonin 2C receptor (gene name htr2c) relative to surrounding regions such as the VTA, and that microinfusion of a specific 5-HT2CR antagonist into the RMTg significantly attenuated cocaine conditioned avoidance. Because cocaine binds to both serotonin and dopamine uptake transporters, I postulated 5-HT2CR activation at the RMTg might be a novel mechanism by which RMTg activates in response to cocaine and contributes to cocaine?s aversive effects. In this proposal, I will definitively test the hypothesis that 5-HT2CR activation facilitates RMTg excitation, and that the 5-HT2CR is necessary for RMTg response to cocaine and cocaine conditioned avoidance and relapse. In specific aim 1, to determine the effect of 5-HT2C receptor activation on VTA-projecting RMTg neurons, I will use in vitro slice electrophysiology techniques. In specific aim 2, to determine the role of 5- HT2CR in driving RMTg and behavioral responses to cocaine, I will use AAV mediated shRNA to knock down 5- HT2CR expression at the RMTg in vivo while measuring cocaine-induced RMTg activation and behavioral responses. If successful, these studies will provide the first insight into the role of serotonin receptors in the modulation of RMTg activity, identify a novel role of 5-HT2C receptor at the RMTg that acts in concert with LHb inputs to mediate cocaine?s aversive effects, and identify 5-HT2CR antagonism to be a novel and valid strategy for treating cocaine?s aversive effects and addiction.